Heterotrimeric G-protein subunit function in Candida albicans: both the alpha and beta subunits of the pheromone response G protein are required for mating

Abstract

A pheromone-mediated signaling pathway that couples seven-transmembrane-domain (7-TMD) receptors to a mitogen-activated protein kinase module controls Candida albicans mating. 7-TMD receptors are typically connected to heterotrimeric G proteins whose activation regulates downstream effectors. Two Galpha subunits in C. albicans have been identified previously, both of which have been implicated in aspects of pheromone response. Cag1p was found to complement the mating pathway function of the pheromone receptor-coupled Galpha subunit in Saccharomyces cerevisiae, and Gpa2p was shown to have a role in the regulation of cyclic AMP signaling in C. albicans and to repress pheromone-mediated arrest. Here, we show that the disruption of CAG1 prevented mating, inactivated pheromone-mediated arrest and morphological changes, and blocked pheromone-mediated gene expression changes in opaque cells of C. albicans and that the overproduction of CAG1 suppressed the hyperactive cell cycle arrest exhibited by sst2 mutant cells. Because the disruption of the STE4 homolog constituting the only C. albicans gene for a heterotrimeric Gbeta subunit also blocked mating and pheromone response, it appears that in this fungal pathogen the Galpha and Gbeta subunits do not act antagonistically but, instead, are both required for the transmission of the mating signal.

FIG. 1.

Strains with CAG1 deleted are sterile. The mating was assayed by auxotrophic marker complementation with strains of opposite mating types. Cells from opaque colonies were grown at 24°C on a YPD plate and crossed for 24 h before transfer by replica plating onto a plate lacking five amino acids for the selection of the mating products shown here after 5 days of incubation (the YPD template is shown on a reduced scale in the lower part of each panel). (A) Mating assay for MTLa Δcag1 strains and strains with CAG1 reintegrated (Δcag1+CAG1). wt, wild type. (B) Mating assay for MTLα Δcag1 strains and strains with CAG1 reintegrated. No colonies of the Δcag1 strains of both mating types (CA114, CA116, CA119, CA132, and CA137) were detectable, while the strains with CAG1 reintegrated (CA121, CA122, and CA141) reverted the sterile phenotype. Opaque cells from the Δcag1 strains used in the experiment were morphologically similar to the cells of the wild-type parent strain 3294 (data not shown).

FIG. 2.

Δcag1 cells do not respond to the α-factor pheromone. Opaque cells were cultured in the presence of α-factor (+ pheromone) as described in Materials and Methods. Cells from the MTLa strain 3294 responded to the pheromone with the typical formation of unconstricted projections (shmoos). However, no morphological change was observed for the Δcag1 cells from the strain CA114. Similar results were also obtained for the other Δcag1 strain, CA116 (data not shown). Cells from the strains with CAG1 reintegrated (Δcag1+CAG1; CA121 and CA122) had a response similar to that of the wild-type (wt) parent strain 3294 (data not shown for the CA122 strain). Typical shmoos are highlighted with black arrows. The pictures were taken after 6 h of incubation with the pheromone. Similar results were also obtained after 4 h of incubation with the pheromone (data not shown).

FIG. 3.

No genes in MTLa Δcag1 and Δste4 strains are induced by α-factor. DNA microarrays were used to determine the transcription profiles of the strains in the opaque phase after 2 h of incubation in the presence of the α-factor peptide. This figure presents a comparative list of the genes (no name is given for unannotated genes) with a signal ratio—relative to the signal from uninduced cells—greater than 2 (P < 0.05) under at least one condition. The Δste4 CA92 and the Δcag1 CA114 strains showed no induction of the genes induced in the wild-type (wt) 3294 strain. The induction of these genes by the pheromone was at least partially restored in the strains with reintegrated genes, CA104 and CA121. The number of biological replicates in this experiment is indicated by n. Δste4+STE4, Δste4 strain with STE4 reintegrated; Δcag1+CAG1, Δcag1 strain with CAG1 reintegrated.

FIG. 4.

Δste4 cells do not respond to the α-factor pheromone. Opaque cells were cultured in the presence of α-factor (+ pheromone) as described in Materials and Methods. No projections (shmoos) from the MTLa Δste4 strain (CA92) were detected. Shmoo formation was restored in the strain with STE4 reintegrated (Δste4+STE4; CA104). Pictures were taken after 6 h of incubation, and black arrows highlight typical shmoos.

FIG. 5.

Δste4 strains are sterile. The mating assay was done as described for the Δcag1 strains in the legend to Fig. 1. No prototrophic colonies from the Δste4 strains of both mating types (CA26, CA92, and CA100) were detected after 5 days incubation. The reintegration of a copy of the wild-type STE4 gene (Δste4+STE4; strains CA49 and CA104) resulted in the reversion of the sterile phenotype. wt, wild type.

FIG. 6.

Halo assay of MTLa strains overexpressing CAG1. Cells in the opaque phase were spread onto an SD plate. The black dots indicate the positions where 5 μl of either the α-factor synthetic peptide (1 μg/μl; α) or the solvent, as a negative control (control), was spotted. The plates are shown after 2 days of incubation at 24°C. Halos delineate the zones of growth arrest induced by α-factor. The overexpression of the CAG1 gene (o/e CAG1) suppresses the sensitivity of the Δsst2 strain to the pheromone. wt, wild type.